Gel electrophoresis is an extremely versatile tool with which to identify compounds associated with developments in biotechnology. Electrophoresis is used extensively for the separation, identification and preparation of pure samples of nucleic acids, polypeptides, and carbohydrates. In many applications, one wishes to separate components of a mixture, where the components may vary in relatively subtle ways. Separations may be associated with the detection of biomolecules (polypeptides, polynucleotides, polysaccharides, and combinations thereof, for example, glycosylated proteins, DNA-protein complexes) having different molecular characteristics, such as numbers of monomers, different sequences, different conformations, different charge/mass ratios, or different hydrophobicities/hydrophilicities. Essential to the success of the gel electrophoresis is the nature of the gel and the manner in which it is prepared.
The gel medium in electrophoresis serves one or more functions. For example, the medium can serve as an anti-convective support, a molecular sieve, a gradient of pH, or some other function. For the most part, two compositions dominate the gel compositions which are generally employed in slab gel formats: polyacrylamide and agarose. The polyacrylamides are normally crosslinked to provide for a sieving structure, where the proportion of crosslinking monomers determines the molecular weight range which may be separated by the gel. The addition polymers are normally formed in situ, where substantial care must be taken in the preparation of the gel to insure uniformity, substantial completion of the polymerization, and reproducibility of the separations achieved on the gel. For agarose, the source of agarose is a naturally occurring material, so there can be great variation in the quality of the agarose, the nature of the contaminants, and the like. Therefore, there is substantial uncertainty in going from one batch to another batch of agarose whether one is obtaining gels of comparable quality.
While the polyacrylamides used in slab gel electrophoresis are normally crosslinked, this crosslinking is not required for molecular sieving, but rather for providing anticonvective and mechanical properties to the medium. By contrast, in capillary electrophoresis, mechanical integrity and anticonvective properties are not requirements of the gel medium and un-crosslinked solutions are commonly used for their molecular sieving properties. Barron (1995) reviews the prior art with respect to the large variety of un-crosslinked polymer solutions that have been used in capillary electrophoresis, including hydroxyethylcellulose, polyacrylamide, and various other polymers. Un-crosslinked polymers have been used in slab gel electrophoresis when the un-crosslinked polymer is contained in a composite matrix with agarose. In this manner, the agarose contributes mechanical and anticonvective properties, and prevents the un-crosslinked polymer from dissolving into the surrounding buffer. Examples include combinations of un-crosslinked polyacrylamide and agarose (Bode et al.) and combinations of hydroxyethylcellulose and agarose (Perlman et al.).
Major shortcomings remain in current methods of using un-crosslinked polymers in capillary and slab gel electrophoresis. Even moderate concentration solutions of high molecular mass polymers are very viscous, and difficult to load in narrow bore capillaries or to pour in slab gel chambers. The viscosity of un-crosslinked polyacrylamide in aqueous solution demonstrates a log-linear temperature profile, i.e. the log of the viscosity decreases linearly with temperature. This behavior is typical of hydrophilic polymers, and is found in solutions of hydroxyethylcellulose, polyethylene oxide and other hydrophilic, water-soluble polymers. Thus, the viscosity of these solutions is reduced by an equal percentage for every unit increase in temperature, thereby requiring large temperature increases to obtain sufficient viscosity reduction.
The preparation and formation of the gel is not simple and there are many problems in insuring that bubble formation does not occur, that there are no hot spots, and there is homogeneity. While filling a slab gel is an art form, filling a capillary for capillary electrophoresis is a matter of greater complexity. Furthermore, despite the complexity and difficulties in preparing the gels, both slab and capillary, one may only be able to use the gel once and then have to discard it. This means that a substantial proportion of time for obtaining the result from gel electrophoresis is involved with the preparation of the gel.
One of the reasons there are so few materials which have found acceptance for gels is the relatively large number of parameters which the gels must fulfill. Included among these parameters are excellent resolution, handling properties, optical clarity, mechanical strength, ease of transfer of separated sample to other substrates, acceptance of various reagents for binding to or reacting with the bands of the sample, and ease of recovery of the sample. To retain all of these characteristics at a high performance level while still improving other characteristics, such as ease of forming the gel slab or gel containing electrophoretic capillary and ease of handling during the formation of the gels in the holder, has proved to be extremely elusive.
Alternative compositions to those described above have been suggested such as combinations of agarose and galactomannan (U.S. Pat. No. 5,230,832), modified cellulose (Perlman et al. Anal. Biochem. 163:247-254 (1987)), water soluble gums (U.S. Pat. No. 4,290,911; 4,894,250; and 4,952,686), and 1,3-glucans (WO 93/08200). Hydroxyethylcellulose has been suggested for use in capillary electrophoresis (Barron et al. J. Chromatography A, 652:3-16 (1993)). Entangled polymer solutions for capillary electrophoresis have been described in Heller, J. Chromatography A, V710N2 (1995) 309-32 1.